Purification and Characterization of a Fibrinolytic Enzyme from Bacillus pumilus 2.g Isolated from Gembus, an Indonesian Fermented Food

Abstract

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than 60°C. Fibrinolytic activity was increased by 5 mM MgCl2 and 5 mM CaCl2 but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the α and β chains of fibrinogen but was unable to degrade the γ chain.

Keywords: Bacillus pumilus; fermented food; fibrinolytic enzyme; gembus.

Fig. 1

Changes in the growth (A) and fibrinolytic activities (B) of B. pumilus 2.g cultured in different growth media.

Fig. 2

SDS-PAGE (A) and fibrin zymography (B) of culture supernatant from B. pumilus 2.g incubated in NB medium. Lane M, broad-range size marker (Dokdo-Mark™, ElpisBiotech, Daejon, Korea); lanes 1~8: culture supernatant from 12 h (1), 24 h (2), 36 h (3), 48 h (4), 60 h (5), 72 h (6), 84 h (7), and 96 h (8) of incubation.

Fig. 3

Elution profile of a fibrinolytic enzyme from B. pumilus 2.g through a CM-Sephadex column (A) and a Phenyl Sepharose 6-FF column (B).

Fig. 4

SDS-PAGE (A) and fibrin zymography (B) of protein samples at different purification stages. Lane M, size marker (Dokdo-Mark™); lanes 1~3: sample after 80% ammonium sulfate precipitation (1), sample after CM-Sephadex purification (2), sample after Phenyl Sepharose 6-FF purification (3).

Fig. 5

Effect of pH (A) and temperature (B) on the stability of a partially purified fibrinolytic enzyme from B. pumilus 2.g.

Fig. 6

Fibrinogen hydrolysis by a partially purified enzyme from B. pumilus 2.g. Lane M, size marker (Dokdo-Mark™); lane 1: control (no enzyme treatment); lanes 2~9: fibrinogen after treatment with purified enzyme for 0 min (2), 10 min (3), 30 min (4), 1 h (5), 2 h (6),4 h (7), 8 h (8), and 12 h (9).